Sequence variation of commercially available kratom products at universal DNA barcode regions.

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a 'legal high' and has gained popularity as an alternative to opioids. However, kratom's increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.

Development of a novel five dye insertion/deletion (INDEL) panel for ancestry determination.

The use of genetic markers, specifically Short Tandem Repeats (STRs), has been a valuable tool for identifying persons of interest. However, the ability to analyze additional markers including Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (INDELs) polymorphisms allows laboratories to explore other investigative leads. INDELs were chosen in this study because large panels can be differentiated by size, allowing them to be genotyped by capillary electrophoresis. Moreover, these markers do not produce stutter and are smaller in size than STRs, facilitating the recovery of genetic information from degraded samples. The INDEL Ancestry Informative Markers (AIMs) in this study were selected from the 1000 Genomes Project based on a fixation index (FST) greater than 0.50, high allele frequency divergence, and genetic distance. A total of 25 INDEL-AIMs were optimized and validated according to SWGDAM guidelines in a five-dye multiplex. To validate the panel, genotyping was performed on 155 unrelated individuals from four ancestral groups (Caucasian, African, Hispanic, and East Asian). Bayesian clustering and principal component analysis (PCA) were performed revealing clear separation among three groups, with some observed overlap within the Hispanic group. Additionally, the PCA results were compared against a training set of 793 samples from the 1000 Genomes Project, demonstrating consistent results. Validation studies showed the assay to be reproducible, tolerant to common inhibitors, robust with challenging casework type samples, and sensitive down to 125 pg. In conclusion, our results demonstrated the robustness and effectiveness of a 25 loci INDEL system for ancestry inference of four ancestries commonly found in the United States.

The development of a next-generation sequencing panel targeting cannabinoid synthase genes to distinguish between marijuana and hemp.

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n = 147).

Development of a novel five-dye panel for human identification insertion/deletion (INDEL) polymorphisms.

DNA analysis of forensic case samples relies on short tandem repeats (STRs), a key component of the combined DNA index system (CODIS) used to identify individuals. However, limitations arise when dealing with challenging samples, prompting the exploration of alternative markers such as single nucleotide polymorphisms (SNPs) and insertion/deletion (INDELs) polymorphisms. Unlike SNPs, INDELs can be differentiated easily by size, making them compatible with electrophoresis methods. It is possible to design small INDEL amplicons (<200 bp) to enhance recovery from degraded samples. To this end, a set of INDEL Human Identification Markers (HID) was curated from the 1000 Genomes Project, employing criteria including a fixation index (FST) ≤ 0.06, minor allele frequency (MAF) >0.2, and high allele frequency divergence. A panel of 33 INDEL-HIDs was optimized and validated following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, utilizing a five-dye multiplex electrophoresis system. A small sample set (n = 79 unrelated individuals) was genotyped to assess the assay's performance. The validation studies exhibited reproducibility, inhibition tolerance, ability to detect a two-person mixture from a 4:1 to 1:6 ratio, robustness with challenging samples, and sensitivity down to 125 pg of DNA. In summary, the 33-loci INDEL-HID panel exhibited robust recovery with low-template and degraded samples and proved effective for individualization within a small sample set.

Comparison of nine extraction methods for bacterial identification using the ONT MinION sequencer.

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was only obtained in 63% of the cases.

Optimization of InnoXtract™ extraction and purification system for DNA extraction from skeletal samples.

The InnoXtract™ extraction and purification system is a purification method designed for DNA extraction from low-template samples, specifically rootless hair shafts. Its ability to successfully capture highly fragmented DNA suggests its suitability for use with other challenging sample types, including skeletal remains. However, the lysis and digestion parameters required modifications to successfully optimize the method for this sample type. A two-part digestion was developed utilizing a homebrew digestion buffer (0.5 M EDTA, 0.05% Tween 20, and 100 mM NaCl) and a supplemental lysis with the Hair Digestion Buffer included in the InnoXtract™ kit. Additionally, the magnetic bead volume was modified to improve DNA recovery from these challenging samples. With the altered protocol, the quality and quantity of DNA recovered from InnoXtract™ extracts were comparable to another commercial skeletal extraction method (PrepFiler™ BTA). This modified extraction method successfully purified sufficient amounts of quality DNA from a variety of skeletal samples to produce complete STR profiles. Successful STR typing from surface decomposition, burned, cremated, buried, and embalmed remains indicates the potential of this new method for challenging human identification and missing-person cases.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

Detection and analysis of DNA mixtures with the MiSeq FGx®.

Recognizing and interpreting mixtures are challenges that occur frequently in forensic casework. Therefore, any new analysis methods that are implemented must handle the challenges of mixed forensic samples. Next generation sequencing offers advantages over capillary electrophoresis in amplicon multiplexing and degraded sample analysis; however, advantages with mixed samples rely heavily on the advancement of user-friendly analysis software. This research analyzed samples with the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx® and compared them with the GlobalFiler™ STR Kit for capillary electrophoresis. Metrics tested for both chemistries included concordance, limits of detection, and mixture analysis. Data analysis for mixture samples was completed with the MixtureAce™ plug-in and ArmedXpert™ software. Next generation sequencing offered distinct advantages in limits of detection and isoallele heterozygosity but suffered from increased variability in stutter and allele count ratios compared to capillary electrophoresis.

Evaluation of chloroplast DNA barcoding markers to individualize Papaver somniferum for forensic intelligence purposes.

The opium poppy, Papaver somniferum L., is a forensically important plant due to the medicinal and illegal uses for the milky latex stored in the pods. This latex contains the alkaloids morphine, codeine, and thebaine that are used for their analgesic properties and/or for synthesizing other opioids. However, these compounds are highly addictive and have caused a national opioid epidemic. Two other Papaver species, P. setigerum DC. and P. bracteatum Lindl., are also of forensic interest because they pose both forensic and legal issues. They are largely uncontrolled under the Controlled Substances Act, making these species a common defense strategy. Current morphological and chemical identification methods have been moderately successful but have drawbacks. There is also a lack of sequencing data available. Therefore, exploiting the genome using chloroplast DNA barcoding markers could help to accurately identify these species of interest when plant material is taken. This study screened and assessed the genetic variation both between species and within populations of P. somniferum in nine cpDNA barcode regions (ndhF-rpl32, petA-psbJ, rpl32-trnL, rps16-trnQ, trnE-trnT, trnH-psbA, trnL-trnF, rpl16 intron, and psbE-petL). Published reference genomes from the NCBI GenBank database were aligned and compared for an initial in silico screening. Additionally, ten P. somniferum seed samples from various vendors were sequenced and compared across samples and to published reference data at the various barcode regions of interest. This study showed that the regions trnH-psbA and petA-psbJ have promise for utility in individualization for both inter- and intra-species individualization of P. somniferum.

Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies.

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.

Evaluation of tetrahydrocannabinolic acid (THCA) synthase polymorphisms for distinguishing between marijuana and hemp.

The Controlled Substances Act (CSA) classifies marijuana (Cannabis sativa) as a Schedule I illicit drug. However, the recent Agriculture Improvement Act of 2018 (U.S. Farm Bill) removed hemp from the definition of marijuana in the CSA, making it a legal crop. As a result, many hemp products are now available, including strains of hemp buds high in other cannabinoids such as cannabidiol (CBD) or cannabigerol (CBG). The genetic inheritance of chemical phenotype (chemotype) has been widely studied, with the tetrahydrocannabinolic acid (THCA) synthase gene at the forefront. Previous studies have speculated that there are two forms of the THCA gene, one that produces an active enzyme (present in marijuana) and one that cannot produce a functional enzyme (present in hemp). A DNA analysis method is desirable for determining crop type in sample types inconducive to chemical analysis, such as immature crops, trace residues, small leaf fragments, seeds, and root material. This study optimized and evaluated a previously reported single nucleotide polymorphism (SNP) assay for determining C. sativa crop type. Furthermore, the presence or absence of 15 cannabinoids, including THC and THCA, was reported in cannabis reference materials and 15 legal hemp flower samples. The SNP assay correctly identified crop type in most samples. However, several marijuana samples were classified as hemp, and several hemp seeds were classified as marijuana. Two strains of legal CBG hemp flowers were also classified as marijuana, indicating that factors other than the genetic variation of the THCA synthase gene should be considered when determining crop type.

Massively parallel sequencing of Cannabis sativa chloroplast hotspots for forensic typing.

BACKGROUND: Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA, and the use of DNA barcodes could assist drug trafficking investigations by indicating the biogeographical origin and crop type of a sample and providing a means for linking cases. Additionally, the legality of marijuana in the USA remains complicated with some states fully legalizing marijuana for recreational use while federally marijuana remains completely illegal. Massively parallel sequencing (MPS) offers distinct advantages over capillary electrophoresis (CE), including more comprehensive coverage of target loci, analysis of hundreds of markers simultaneously, and high throughput capabilities. METHODS: This study reports on the development of a MiSeq FGx® assay targeting seven "hotspot" regions in the Cannabis sativa chloroplast genome that are highly polymorphic and informative in attempts to determine biogeographical origin and distinguishing between marijuana and hemp. Sequencing results were compared to previous studies that used CE-based genotyping methods. RESULTS: A total of 49 polymorphisms were observed, 16 of which have not been previously reported. Additionally, sequence data revealed isoalleles at one locus, which were able to differentiate two samples that had the same haplotype using CE-based methods. This study reports preliminary results from sequencing 14 hemp and marijuana samples from different countries using the developed MPS assay. CONCLUSION: Future studies should genotype a more comprehensive sample set from around the world to build a haplotype database, which could be used to provide investigative leads for law enforcement agencies investigating marijuana trafficking.

Comparison of DNA extraction techniques for the recovery of bovine DNA from fly larvae crops.

Forensic entomology aids investigations using insects and is primarily associated with the estimation of post-mortem interval (PMI). Studies have shown that human DNA can be recovered from the crops of fly larvae. While several factors regarding the recovery of human DNA from crops have been studied, DNA extraction methods have not been thoroughly assessed. Determining a method for optimal extraction could aid crime laboratories in implementing DNA extraction from larvae and streamlining future research. Bovine DNA was used as a substitute for human DNA to test several DNA extractions kits. Four DNA extraction kits (Chelex®, PDQeX forensicGEM, EZ1® DNA Investigator, DNeasy® Powersoil® Pro Kit) were evaluated based on the quantity and quality of bovine DNA extracted. Extractions were performed on whole fly larvae and dissected crops. Quantification was performed using real-time PCR (qPCR) on a StepOne™ Real-Time PCR System with SYBR® Green using bovine-specific cytochrome b primers. The quality of extracts was determined by checking for inhibition using commercial qPCR chemistries with an internal PCR control (IPC). When using whole fly larvae, Powersoil® Pro yielded the highest average DNA yield (n = 10, 0.668 ± 0.458 ng/µl), while EZ1® DNA Investigator yielded the highest average with crops (n = 10, 0.605 ± 0.403 ng/µl). Chelex and forensicGEM yielded low amounts of bovine DNA, and its extracts were inhibited, unlike EZ1® and Powersoil® Pro, which have purification steps. Therefore, it is recommended to use EZ1® DNA Investigator coupled with automation on EZ1® Advanced XL to recover DNA from fly larvae crops.

Collection and storage of DVI samples with microFLOQ® Direct swabs for direct amplification.

The rapid identification of decomposing human remains is a crucial component of disaster victim identification (DVI), often occurring in remote areas without access to laboratory or storage facilities. Due to the ease of collection and amenability to storage in harsh conditions, swabs may be used to collect DNA from decomposing remains as an alternative to sampling tissue or bone. Direct amplification could further streamline the process and reduce costs. This study investigated the efficacy of direct amplification of DVI samples using microFLOQ® Direct swabs and the QIAGEN Investigator QS GO! Kit. A comparison of performance between direct amplification and traditional methods was made to assess whether direct amplification offered an improvement to traditional methods. DNA was collected by swabbing the muscle of a decomposing human cadaver using three swab types (ADS Genetics 4N6FLOQSwabs®, NADS Genetics 4N6FLOQSwabs®, and the microFLOQ® Direct swab). Traditional swabs (4N6FLOQSwabs®) were extracted and quantified, while a direct amplification strategy was used with the microFLOQ® Direct swabs coupled with the Investigator 24Plex GO! Kit. Processing of the microFLOQ® Direct swabs were optimized and a hybrid strategy that used 4N6FLOQSwabs® to collect and store DNA before swabbing or "subsampling" the 4N6FLOQSwabs® for processing with microFLOQ® Direct swabs was developed. This hybrid strategy allowed for rapid processing without the consumption of the original sample. Traditional and direct PCR methods were comparable up to day 10 of decomposition depending on the sample location and for up to 3 months of storage at room temperature. This research indicated that microFLOQ® Direct swabs in conjunction with the Investigator 24Plex GO! Kit can be used to facilitate rapid direct processing of DNA from decomposing human remains.

The performance of quality controls in the Investigator® Quantiplex® Pro RGQ and Investigator® 24plex STR kits with a variety of forensic samples.

Forensic DNA laboratories process database reference samples on FTA® cards or buccal swabs, which commonly contain adequate amounts of quality DNA resulting in full STR profiles and high first-pass rates. However, some reference samples and many forensic casework samples are exposed to a variety of insults that may lead to low quantities of DNA, DNA degradation, DNA mixtures, and/or PCR inhibition, posing a challenge to downstream genotyping success. The inclusion of multiple amplification targets and internal PCR controls (IPCs) in DNA quantification kits, and quality sensors within STR amplification kits can aid in the accurate interpretation of sample/profile quality, and guide more efficient rework strategies when needed. In order to assess the effectiveness of these quality systems we subjected database-type samples (buccal swabs and blood or saliva on FTA® cards), mock casework samples (low-template, degraded, inhibited, DNA mixtures), and authentic post-coital samples to various challenging conditions. Concordance between the quality flags in the Investigator® Quantiplex® Pro RGQ kit (QIAGEN), the QS markers in QIAGEN's Investigator® 24plex QS kit, and overall STR profile quality was evaluated for all casework-type samples. To assess the value of the QS markers in the Investigator® 24plex QS and GO! STR kits, samples with partial or failed STR profiles were reworked based on the quality of the electropherogram first with the QS markers redacted, and second in conjunction with the QS markers. Results from each of the rework approaches were compared to determine which strategy, if any, improved the STR profile quality and the number of reportable alleles. The QS markers in the 24plex STR kits correctly confirmed sample quality in 99.9% of databasing samples and 98% of mock casework samples. Quality flags during DNA quantification were concordant with downstream STR profiles for the majority (77%) of the mock casework samples. Additionally, when samples with partial STR profiles were reworked, more loci were obtained for 80% of the samples regardless of the rework strategy used. However, the most notable improvement in STR completeness was observed in inhibited samples that were reworked based on the information provided by the STR quality sensors, with an average increase of 56% reportable alleles.

Novel extraction chemistry and alternative amplification strategies for use with rootless hair shafts.

Rootless hair shafts are often considered unsuitable for STR genotyping due to the known high failure rate. The same samples can be reliably processed with mitochondrial sequencing. However, the minimal discriminatory power of widely implemented control region mitochondrial sequencing techniques limits its utility in some forensic casework. In this research, multiple variables were tested to provide information on rootless hair shaft sample genotyping success. Results showed external decontamination procedures decreased drop-in alleles but also greatly reduced profile recovery. The novel InnoXtract™ chemistry was comparable to automated EZ1 DNA Investigator extraction. With thoroughly decontaminated hairs, InnoTyper® 21 amplification generated random match probabilities higher than STR chemistry in 71.875% of samples and 18.75% of samples benefitted from the use of InnoTyper® 21 amplification compared with estimated mtDNA profile rarity. Compared with the capillary electrophoresis-based amplification chemistries tested, the ForenSeq™ DNA Signature Prep chemistry paired with massively parallel sequencing was the most discriminatory amplification strategy tested.

Evaluation of the trnK-matK-trnK, ycf3, and accD-psal chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa.

Cannabis sativa (marijuana and hemp) is one of the most controversial crops worldwide. In the USA, the state-specific legalization of marijuana and recently legalized hemp pose a problem for law enforcement. This study seeks to utilize chloroplast hSTRs, INDEL, and SNPs markers to develop genotyping methods to aid in the differentiation of legal hemp from illicit marijuana and also for tracking the flow of trafficked marijuana. Three polymorphic regions: trnK-matK-trnK, ycf3, and accD-psal, of the C. sativa chloroplast genome were evaluated in order to distinguish crop type and biogeographic origin. A total of nine polymorphic sites were genotyped from five distinct populations (hemp from the USA and Canada, marijuana from Chile and USA-Mexico, and medical marijuana from Chile) with a custom fragment and SNaPshotTM assay. The study also combined genotype results from the same sample set using 21 additional polymorphic markers from previous studies. The effectiveness of these multi-locus assays to distinguish sample groups was assessed using haplotype analysis, phylogenetic analysis, pairwise comparisons, and principal component analysis. Results indicated a clear separation of Canadian hemp using only the nine polymorphic sites developed in this study. The additional 21 markers were able to separate US hemp from both marijuana groups to a significant level (p < 0.05) when assessing average Fixation Indices (FST). This study demonstrated the applicability of these organelle markers for the determination of crop type and biogeographic origin of C. sativa. However, a more extensive database is needed to evaluate the true discriminatory power of these markers.

Use of Eucalyptus DNA profiling in a case of illegal logging.

Eucalyptus is grown world-wide for paper pulp, solid wood, and other industries. Theft or illegal cutting of the trees causes hardship to owners of plantations and countries whose economies rely on the sale and export of eucalyptus products. Unfortunately, many of these crimes go unpunished due to lack of forensic evidence. Over 1200 short tandem repeat (STR) markers have been identified in the genomes of genus Eucalyptus and related species. However, their importance and utility in aiding forensic investigations of wood theft have not been explored. This study evaluated nine STRs for diversity and applied them to a case involving suspected wood theft. As expected, three dinucleotide STR markers showed greater variability but resulted in harder to interpret profiles. Four STR tetranucleotide markers evaluated in this study were found to contain additional repeat structures (dinucleotide or trinucleotide) that enhanced their variability but resulted in profiles with peaks at multiple stutter positions and heterozygote peak imbalance. The most promising STR markers were EGM37 and EMBRA 1374. Though less variable, they yielded robust and reproducible DNA profiles. All nine STR markers were applied to a case involving suspected wood theft. Samples were collected from seized wood and from remaining stumps in a plantation. No DNA match was found, thus eliminating the evidence samples as having originated from the forest. Dendrochronology analysis also resulted in an exclusion. This case study represents the first report using STR markers in any eucalyptus species to provide DNA evidence in a case of suspected wood theft.

Investigation of chloroplast regions rps16 and clpP for determination of Cannabis sativa crop type and biogeographical origin.

Cannabis sativa can be classified as either hemp (a legal crop containing less than 0.3% delta-9-tetrahydrocannabinol, THC) or marijuana (an illegal drug containing more than 0.3% THC). Despite its legalization in 33 states for medicinal or recreational use, marijuana remains the most commonly used illicit drug in the USA, and it is heavily trafficked into and within the country. Discriminating between marijuana and hemp is critical to the legal process. Genetic analysis provides a means of analyzing samples unsuitable for chemical analysis, and in addition to discriminating between crop types, DNA may be able to determine the biogeographical origin of samples. In addition, the sharing of rare haplotypes between different seizures may be useful for linking cases and providing investigative leads to law enforcement. This study evaluates the potential of two highly polymorphic regions of the chloroplast genome of C. sativa, rps16 and clpP, to be used for determination of crop type and biogeographical origin. Custom fragment analysis and SNaPshot™ assays were developed to genotype nine polymorphic loci in hemp samples from the USA and Canada, marijuana samples from USA-Mexico and Chile, and medical marijuana samples from Chile. Haplotype analysis revealed eight haplotypes. Only Canadian hemp could be completely differentiated from the other sample groups by haplotype. Phylogenetic analysis and principal component analysis suggested a closer relationship among USA-Mexico marijuana, Chilean marijuana and medical marijuana, and USA hemp. Genotyping additional polymorphisms in future studies is expected to reveal further differences between these sample groups.